Project Summary Alzheimer's disease (AD) creates substantial human suffering and social financial burdens. At present, the search for effective disease-modifying AD treatments is substantially hampered by our incomplete understanding of the cellular mechanisms and pathways that change in human neurons during AD. Here we propose to substantially increase our understanding of these problems by testing an overarching hypothesis tying variation in Amyloid Precursor Protein (APP) transcytotic trafficking to pathways that lead to AD. Specifically, we propose to test the hypothesis that one major contributor to the molecular and cellular complexity of AD derives from variability in branches of pathways controlling trafficking of APP and its fragments to the neuronal axon. This hypothesis emerges in part from the likely endocytic nature of a number of functions identified by GWAS and in part from our recent work on neuronal trafficking defects in a series of FAD mutations we generated and analyzed using human induced pluripotent stem cell (hIPSC) technology. We also propose to increase our understanding of the uniquely neuronal endocytic and transcytotic pathways mediating APP trafficking to neuronal axons. This trafficking is key to the anatomical location of A? secretion, plaque formation, APP proteolytic processing and the control of axon-specific tau phosphorylation leading to the formation of pathogenic tau aggregates called neurofibrillary tangles (NFT). Thus, we also propose to test a unique model we have developed that describes two different pathways for APP entry into neuronal axons, one of which we suggest controls axonal tau phosphorylation. We will then extend this work to study FAD and AD-protective APP mutations and their interaction with the traffic pathways of APP to the axon. The proposed three specific aims are: 1) Test the hypothesis that there are two somatodendritic pathways that traffic APP to neuronal axons. 2) Test the mechanistic hypothesis that direct interactions of APP and SORLA mediate transcytotic functions that modulate axonal entry and amyloidogenic processing of APP. 3) Test the hypothesis that ESCRT- functions control endo-lysosomal trafficking of APP and subsequent transcytotic amyloidogenic processing and axonal entry. Collectively, the proposed experiments will provide mechanistic insights into the various branches of APP trafficking which are critical for amyloidogenic APP processing and phospho-tau levels, opening up new avenues in AD research and identification of potential targets for therapeutic intervention.